Peroxynitrite induces tyrosine residue modifications in synaptophysin C-terminal domain, affecting...

Department of Cell Biology and Neuroscience, Istituto Superiore di Sanita, Viale Regina Elena, Rome, Italy

Neurotransmitter release occurs through a complex series ofsynaptic vesicle (SV) trafficking steps, including dockingand fusion with pre-synaptic plasma membrane followed byrecycling of the SV membrane by endocytosis (Sudhof2004). Synaptophysin (SYP) is a major integral membraneprotein of SVs (Jahn et al. 1985), and is widely expressedthroughout the nervous system (Marqueze-Pouey et al.1991), in chromaffin and neurosecretory granules (Schillingand Gratzl 1988). SYP shares with its homologous synap-toporin and pantophysin a common transmembrane topology,with four membrane-spanning domains and cytoplasmicamino- and carboxy-terminal regions (Hubner et al. 2002).Based on its predicted structure, it has been suggested thatSYP forms homo-oligomers in vitro (Thomas et al. 1988) aswell as in vivo (Pennuto et al. 2002). This has led to thesuggestion that SYP may participate in fusion pore formationduring neurotransmitter release (Gincel and Shoshan-Bar-matz 2002; Arthur and Stowell 2007). The assembly ofhomo-oligomers may also facilitate cholesterol accumulationin the plane of the plasma membrane, thus driving SYP ontothe nascent vesicle and contributing to the recruitment of

other SV proteins (Thiele et al. 2000). Indeed, SYP interactswith other synaptic terminal proteins, including vesicle-associated membrane protein (VAMP2), also known assynaptobrevin 2 (Edelmann et al. 1995; Felkl and Leube2008), the vesicular proton pump V-ATPase (Galli et al.1996), myosin V (Prekeris and Terrian 1997), dynamin I(Daly et al. 2000), and c-adaptin (Horikawa et al. 2002).

Received May 6, 2009; revised manuscript received August 4, 2009;accepted August 27, 2009.Address correspondence and reprint requests to Anna Maria Michela

Di Stasi, Istituto Superiore di Sanita, Department of Cell Biology andNeuroscience, Viale Regina Elena, Rome 299 – 00161, Italy.E-mail: [email protected] used: aa, amino acids; ECL, enhanced chemilumines-

cence; GST, glutathione-S-transferase; LC–MS/MS, liquid chromatog-raphy tandem mass spectrometry; MALDI, matrix-assisted laserdesorption/ionisation; NO2Y, nitrotyrosine; pY, phosphotyrosine; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis;SH, src homology; SV, synaptic vesicle; SYP, synaptophysin; TBS, Tris-buffered saline ; ToF, time of flight; TTBS, TBS containing 0.05%Tween 20; VAMP2, vesicle-associated membrane protein.

Abstract

Peroxynitrite is a potent oxidant that contributes to tissue

damage in neurodegenerative disorders. We have previously

reported that treatment of rat brain synaptosomes with per-

oxynitrite induced post-translational modifications in pre- and

post-synaptic proteins and stimulated soluble N-ethylmalei-

mide sensitive fusion proteins attachment receptor complex

formation and endogenous glutamate release. In this study we

show that, following peroxynitrite treatment, the synaptic

vesicle protein synaptophysin (SYP) can be both phosphory-

lated and nitrated in a dose-dependent manner. We found that

tyrosine-phosphorylated, but not tyrosine-nitrated, SYP bound

to the src tyrosine kinase and enhanced its catalytic activity.

These effects were mediated by direct and specific binding of

the SYP cytoplasmic C-terminal tail with the src homology 2

domain. Using mass spectrometry analysis, we mapped the

SYP C-terminal tail tyrosine residues modified by peroxynitrite

and found one nitration site at Tyr250 and two phosphorylation

sites at Tyr263 and Tyr273. We suggest that peroxynitrite-

mediated modifications of SYP may be relevant in modulating

src signalling of synaptic terminal in pathophysiological con-

ditions.

Keywords: nitrotyrosine, peroxynitrite, phosphotyrosine, src

homology 2 domain, src tyrosine kinase, synaptophysin.

J. Neurochem. (2009) 111, 859–869.

JOURNAL OF NEUROCHEMISTRY | 2009 | 111 | 859–869 doi: 10.1111/j.1471-4159.2009.06378.x

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 859–869 859

How the various interactions of SYP are regulated is stillunclear.

The SYP C-terminal cytoplasmic domain contains ninepentapeptide repeats, each starting with a tyrosine residue thatmay be the target of tyrosine kinases within the nerve terminal.Indeed, SYP is the major tyrosine-phosphorylated protein ofSVs (Pang et al. 1988). SYP is phosphorylated by the non-receptor tyrosine kinase src in vitro (Barnekow et al. 1990),but the functional role of phosphorylation is still unknown(Evans and Cousin 2005). It is temping to speculate thattyrosine phosphorylation of SYPmay regulate its associations,as has been suggested for the interaction with dynamin I, giventhat the tyrosine-based pentapeptide motifs are confined todynamin I binding sites (Daly and Ziff 2002).

Among the kinases of the src family expressed in the CNS(Salter 1998), src and fyn have been shown to play importantroles in synaptic transmission and plasticity at the excitatorysynapses (Kalia et al. 2004). Despite the relative abundanceof src family kinases in the nerve terminals and theidentification of src phosphorylated substrates in SVs (Panget al. 1988; Barnekow et al. 1990; Onofri et al. 1997), littleis known of the functional role and regulation of src kinasesat the pre-synaptic level. Src is enriched in purified SVs andaccounts for the majority of endogenous SV-associatedtyrosine kinase activity (Onofri et al. 1997).

In addition to being a tyrosine-phosphorylated protein,SYP is also nitrated in tyrosine residues when synaptosomesare treated with peroxynitrite, as we have previously shown(Di Stasi et al. 1999). Peroxynitrite is a potent oxidising andnitrating species formed in a diffusion-limited radical–radicalreaction between nitric oxide and superoxide anion (Beck-man et al. 1992). Peroxynitrite-mediated nitration of targetproteins occurs mainly in tyrosine and tryptophan residuesand, in the case of tyrosine, produces 3-nitrotyrosine (3-NO2Y) as major product (Radi 1998). The nitration ofprotein tyrosine residues has been demonstrated underphysiological conditions and it has been shown to increasesignificantly in inflammatory conditions and degenerativediseases (Ischiropoulos and Beckman 2003). Because 3-NO2Y are poor substrates for tyrosine kinases, the nitrationof a tyrosine residue may down-regulate phosphotyrosine(pY)-dependent signalling (Kong et al. 1996). However,several groups, including ours, have reported that peroxyni-trite up-regulates the activity of some src family kinases(Jope et al. 2000) in human pancreatic tumour tissues(MacMillan-Crow et al. 2000), in erythrocytes and insynaptosomes (Di Stasi et al. 1999; Mallozzi et al. 1999;Minetti et al. 2002).

As SYP is also one of the major substrates of vesicle-associated src tyrosine kinase, we wondered whether perox-ynitrite could affect SYP tyrosine phosphorylation. In thisstudy we found that treatment of synaptosomes withperoxynitrite induced tyrosine phosphorylation of SYP.Tyrosine-phosphorylated SYP specifically interacts with the

src homology (SH) 2 domain, resulting in enhancement ofthe kinase catalytic activity. However, tyrosine-nitrated SYPdoes not interact with the src-SH2 domain and does notaffect src catalytic activity. By mass spectrometry (MS)analysis, we identified the tyrosine residues in the SYP C-terminal tail that are targets of phosphorylation and nitration.Our data suggest that oxidative changes induced by peroxy-nitrite at the nerve terminal can modulate interactions ofSYP with proteins involved in signal transduction and in theexo–endocytosis cycle of SVs.

Materials and methods

MaterialsAntibodies were obtained from the following sources: monoclonal

anti-pY (clone 4G10) and anti-NO2Y (monoclonal and polyclonal)

from Upstate Biotechnology (Lake Placid, NY, USA); anti-SYP

(monoclonal and polyclonal) and polyclonal anti-synapsin I from

Synaptic System (Gottingen, Germany); monoclonal anti-src (clone327) from Calbiochem-Oncogene Research Products (Cambridge,

MA, USA). The antibody against glutathione-S-transferase (GST)

was raised in our laboratory, as previously described (Rosa et al.1996). Peroxidase-conjugated goat anti-mouse and goat anti-rabbit

antibodies were purchased from Bio-Rad (Hercules, CA, USA);

partially purified human src [pp60src, purity: 15 U gave a major

band at �60 kDa on a silver-stained sodium dodecyl sulphate–

polyacrylamide gel electrophoresis (SDS–PAGE)] from Upstate

Biotechnology. Immunopure-Protein G and Protein A immobilised

to Trysacryl were purchased from Pierce (Rockford, IL, USA);

nitrocellulose paper from Shleicher and Schuell (Einbeck, Ger-

many). [c32P]ATP (> 3000 Ci/mmol) was obtained from DuPont

NEN (Boston, MA, USA) and Chelex 100 from Bio-Rad; thrombin

from Amersham Biosciences (Uppsala, Sweden). All other reagents

were purchased from Sigma Chemical Co (St Louis, MO, USA).

Synaptosomes preparationAdult male Sprague–Dawley rats (200–250 g) were housed and

handled in accordance with the guidelines laid down by the

European Communities Council directive (86/609/EEC of 24

November 1986) and American Society for Neuroscience. Synap-

tosomes were prepared as previously described (Carlin et al. 1980)with minor modifications. Briefly, brains were homogenised in 10

volumes of Buffer A (0.32 M sucrose, 0.5 mM EGTA and 4 mM

HEPES–NaOH, pH 7.4) in the presence of proteases inhibitors

mixture (Complete� without EDTA; Roche Molecular Biochemi-

cals, Indianapolis, IN, USA) and phosphatases inhibitors 5 mM

NaF, 1 mM Na3VO4, using a Teflon-glass grinder. The homogenate

was centrifuged at 1000 g for 5 min at 4�C to remove nuclei and

debris and the supernatant centrifuged at 13 800 g for 10 min at

4�C. The resulting pellet was resuspended in Buffer A, stratified on

a discontinuous sucrose gradient (0.85, 1 and 1.2 M v/v in HEPES-

buffered sucrose) and centrifuged at 85 000 g for 2 h at 4�C. Thelayer between 1.0 and 1.2 M sucrose (synaptosomal fraction) was

collected, centrifuged at 140 000 g for 20 min, diluted with 4 mM

HEPES–NaOH, pH 7.4 to obtain 0.32 M sucrose concentration and

extensively washed in Buffer A. Protein content was determined by

bicinchoninic acid assay kit (Pierce).

Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 859–869� 2009 The Authors

860 | C. Mallozzi et al.

Peroxynitrite treatmentPeroxynitrite was synthesised as previously described (Radi et al.1991). The concentration of peroxynitrite was determined by

absorbance at 302 nm in 1.5 M NaOH (e302 = 1700/M/cm). Diluted

stock solutions were freshly prepared in 0.1 M NaOH. To avoid

metal-catalysed oxidation of peroxynitrite the phosphate buffer was

treated extensively with Chelex 100 and all samples contained

0.1 mM diethylenetriaminepenta-acetic acid (Beckman et al. 1992).Before peroxynitrite treatment synaptosomes were washed twice in

150 mM phosphate buffer, pH 7.5. Peroxynitrite was added as a

single bolus directly to synaptosomes (10 mg/mL) suspended 1 : 10

in 150 mM phosphate buffer, pH 7.5. After 10 min at 22–25�C,synaptosomes were washed once with phosphate buffer, pH 7.5.

Control experiments were performed with decomposed peroxynitrite

to exclude the participation of contamination products. Decomposed

peroxynitrite was obtained by adding peroxynitrite to the phosphate

buffer for 10 min at 22–25�C before the addition of synaptosomes.

Treatment with 1 mM peroxynitrite was performed in the presence

of 25 mM sodium bicarbonate (at pH 7.5 the CO2 is 1.3 mM) to

enable NO2Y formation (Squadrito and Pryor 1998).

Immunoprecipitation and in vitro kinase assayImmunoprecipitation and in vitro kinase assay was performed as

described (Di Stasi et al. 1999). Synaptosomes were solubilised by

incubation for 30 min at 0�C with an equal volume of 4x

solubilisation buffer [100 mM Tris–HCl, pH 7.5, 0.6 M NaCl, 4%

(wt/vol) Triton X-100, 4% (vol/vol) Na-deoxycholate, 0.4% (vol/

vol) SDS, 0.4 mM Na3VO4, 20 lg/mL leupeptin, 20 lg/mL

aprotinin and 4 mM phenylmethylsulphonylfluoride], diluted twice

with 50 mM Tris–HCl buffer, pH 7.5, 150 mM NaCl (Tris-buffered

saline; TBS) and then centrifuged at 12 000 g for 10 min at 4�C.After pre-clearing with 20 lL of Protein A/G beads prepared as a

50% (vol/vol) suspension for 1 h at 4�C, supernatants were

incubated with the different antibodies and immunocomplexes

precipitated by addition of Protein A or Protein G beads 50% (vol/

vol) suspensions. The immunoprecipitates were collected by

centrifugation and washed twice with solubilisation buffer and

twice with TBS. Bound proteins were eluted with 4x SDS loading

buffer, resolved on SDS–PAGE and analysed by immunoblot with

the specific antibodies. The kinase assay was performed by

incubating the immunoprecipitates in kinase buffer (20 mM Tris–

HCl, pH 7.4, 10 mM MnCl2 and 0.1 mM Na3VO4) containing 1–

2 lCi [c32P] ATP (> 3000 Ci/mmol) for 10 min at 22–25�C. When

indicated an exogenous substrate, such as enolase, was added to the

reaction mixture. The kinase reaction was stopped by adding 4x

SDS loading buffer and the samples were subjected to SDS–PAGE.

The dried gels were exposed to X-ray film for autoradiography. The

kinase activity was measured by autophosphorylation and substrate

phosphorylation using a Phosphor Imager Instrument (Packard,

Camberra, CO, USA).

Gel electrophoresis and immunoblot analysisProteins were separated on 10% SDS–PAGE or using precast 4–

12% 2-(N-morpholino)ethanesulfonic acid-Gels (NuPAGE, novex

high-performance, precast gels from Invitrogen Life Science,

Carlsbad, CA, USA) and proteins were transferred to nitrocellulose

paper. Blots were washed with TBS containing 0.05% Tween 20

(TTBS) buffer and blocked with 3% bovine serum albumin (wt/

vol)–TTBS for 2 h. Washed nitrocellulose filters were incubated

overnight at 4�C with the appropriate antibody. The specificity of

the polyclonal and monoclonal anti-3-NO2Y antibodies was estab-

lished by competition experiments pre-incubating the antibody with

5 mM 3-NO2Y for 10 min, before being added to the blots. This

pre-incubation was sufficient to switch off the enhanced chemilu-

minescence (ECL) signals obtained with both monoclonal and

polyclonal antibodies. After extensive washes in TTBS, the

immunoreactive bands were detected by chemiluminescence cou-

pled to peroxidase activity according to the manufacturer’s

specifications (ECL kit; Pierce).

Recombinant protein expression and purificationDNA coding for the C-terminal cytoplasmic region [amino acids

(aa) 219–308] of SYP was amplified by RT-PCR from mouse brain

total RNA and subcloned into pCRIITOPO (Invitrogen Life

Science). After EcoRI-restriction, SYP insert was subcloned into

pGEX-4T-1 (Amersham Pharmacia Biotech, Piscataway, NJ, USA)

and the construct pGEX-SYP219–307 used to transform BL21 and

TKB1 Escherichia coli cells respectively. To express GST-fused

recombinant protein, cells were incubated 1 h at 37�C in the

presence of 0.5 mM isopropyl-thio-2-D-galactopyranoside. Tyro-

sine-phosphorylated GST–SYP, GST–SYP (pY)B, was expressed in

TKB1 E. coli cells (Stratagene, La Jolla, CA, USA), a strain that

allows to obtain tyrosine-phosphorylated recombinant proteins.

Alternatively, GST–SYP was phosphorylated in vitro by pp60src

(10 U) GST–SYP (pY)src. GST-SH2 domain of src and p85,

subcloned from a mouse cDNA library into pGEX vector, were

kindly provided by Dr. Segatto, IFO-Rome, Italy. GST-fusion

proteins were purified by affinity chromatography on glutathione-

Sepharose beads (Amersham Pharmacia Biotech) following the

manufacturer’s instructions. GST–SYP was nitrated with 1 mM

peroxynitrite as described in ‘Peroxynitrite treatement’ section. GST

was removed from the fusion proteins bound to Sepharose beads by

digestion with thrombin overnight at 4�C following the manufac-

turer’s instructions. For pull-down assay, src-SH2 domains were

biotinylated with sulphosuccinimido-biotin (Sulpho-NHS-biotin)

(Pierce) for 30 min on ice as described (Rosa et al. 1996).

Pull-down experimentsThe GST–SYP, GST–SYP (pY)src, GST–SYP (pY)B, and GST–SYP

(NO2Y) conjugated with glutathione-Sepharose beads were used in

binding assay to recombinant biotinylated-SH2 domains of src or

with synaptosomes. Ten microlitre of bead suspension (5%) were

incubated overnight in a rotating wheel at 4�C with 0.2 and 2 lMSH2 domain in phosphate-buffered saline containing 1% Triton X-

100. The beads were collected by centrifugation and subjected to

extensive washing in binding buffer. The complex was dissociated

adding 4x loading buffer, subjected to SDS–PAGE and transferred

to nitrocellulose. The presence of SH2 domains was detected by

incubating the nitrocellulose with peroxidase-conjugated avidin and

the immunoreactive bands were revealed by ECL. Synaptosomes

were extracted in a buffer (20 mM HEPES, pH 7.4, 50 mM NaCl,

1% Triton X-100), containing a protease inhibitor mixture (Com-

plete� protease inhibitor without EDTA; Roche Molecular Bio-

chemicals) for 1 h at 4�C and then clarified by centrifugation at

15 000 g for 10 min. Synaptosomal extracts (7.5 mg/mL) were

incubated with 50 lg of the indicated GST-fusion proteins immo-

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 859–869

Peroxynitrite affects SYP–src interaction | 861

bilised on 20 lL of glutathione-Sepharose beads overnight at 4�Cunder gentle rotation. After binding, the beads were washed three

times with 1 mL of extraction buffer and boiling in 4x loading

buffer, resolved on SDS–PAGE and eluted bound proteins trans-

ferred to nitrocellulose.

Sepharose-immobilised src-GST-SH2 domain was used in pull-

down experiments of native SYP purified by hydroxyapatite/celite

column, as described in ‘Purification of synaptophysin’ section,

from solubilised synaptosomes treated with 0.1 and 1 mM perox-

ynitrite. Five microlitre of Sepharose beads (25% suspension) were

incubated with native SYP purified from control and peroxynitrite-

treated synaptosomes (�0.1 mg/mL) in phosphate-buffered saline–

1% Triton X-100 (final volume of 200 lL), under rotation overnight

at 4�C. Alternatively, 5 lL of Sepharose beads (25% suspension)

were added to synaptosomal extracts (5 mg/mL) obtained after

solubilisation for 30 min at 0�C in binding buffer (10 mM HEPES,

pH 7.4, 150 mM NaCl, 1% Triton X-100 and 0.1 mM phenylmeth-

ylsulphonylfluoride) and centrifugation at 10 000 g for 10 min.

Then the beads were washed seven times with binding buffer,

solubilised in loading buffer and subjected to SDS–PAGE and

western blot analysis.

Purification of synaptophysinSynaptophysin was solubilised from rat brain synaptosomes

untreated or treated with 0.1 mM and 1 mM peroxynitrite and

purified as described by Gincel and Shoshan-Barmatz (2002) with

some modifications (see Appendix S1).

Identification of phosphorylated and nitrated tyrosine residues bymass spectrometryThe GST–SYP, GST–SYP (pY)B and GST–SYP (NO2Y) were

separated on precast 4–12% 2-(N-morpholino)ethanesulfonic acid-

Gels (NuPAGE) and stained with Coomassie Colloidal Blue

(Invitrogen Life Science). The detectable bands were cut away from

the gel, cut again in small pieces, washed with water and destained

with a solution containing 50 mM ammonium bicarbonate/acetoni-

trile (1 : 1 v/v) (CH3CN; Merck, Darmstadt, Germany). The protein

bands were subsequently subjected to cysteine reduction by 10 mM

dithiothreitol for 1 h at 56�C and alkylation by 50 mM iodoaceta-

mide for 45 min at 20–25�C in the dark, and then were dried by

acetonitrile treatment and finally in a speed vacuum. In-gel digestion

was performed by 12.5 ng/mL endoproteinase AspN (sequencing

grade modified AspN; Promega, Madison, WI, USA) or chymo-

trypsin (Sigma Chemical Co) in 25 mM ammonium bicarbonate at

37�C overnight. To characterise the phosphorylation and the nitration

state of the protein, the peptides mixture coming from AspN

digestion was studied by matrix-assisted laser desorption/ionisation–

time of flight (MALDI–ToF) analysis (see Appendix S1). To

recognise phosphorylated and nitrated sites, peptide mixture from

chymotrypsin incubation was analysed by nanoflow reversed-phase

liquid chromatography tandem MS (LC–MS/MS) using an HPLC

Ultimate 3000 (DIONEX, Sunnyvale, CA, USA) connected on line

with a linear ion trap (ThermoElectron, San Jose, CA, USA).

Peptides have been desalted in a trap column (Acclaim PepMap 100

C18, LC Packings; DIONEX) and then separated in a reverse phase

column, a 10 cm long fused silica capillary (Silica Tips FS 360-75-8;

New Objective, Woburn, MA, USA), slurry-packed in-house with

5 lm, 200 A pore size C18 resin (Michrom BioResources, CA,

USA). Peptides were eluted using a linear gradient from 95%A (H2O

with 5% acetonitrile and 0.1% formic acid) to 60% B (acetonitrile

with 5% H2O with 0.1% formic acid) in 35 min, at 300 nL/min flow

rate. Analyses were performed in positive ion mode and the Spray

Voltage Potential was set up around 1.7 kV. The linear ion trap MS

operated in a data-dependent mode in which each full MS scan was

followed by five MS/MS scans where the five most abundant

molecular ions were dynamically selected and fragmented by

collision-induced dissociation using a normalised collision energy

of 35%. Tandem mass spectra were processed through SEQUEST

algorithm incorporated in BIOWORKS software (version 3.3; Thermo-

Electron). A peptide has been considered legitimately assigned when

it achieved cross-correlation scores of 1.8 for [M + H]1+, 2.5 for

[M + 2H]2+, 3 for [M + 3H]3+, and a probability cut-off for

randomised identification of peptide p < 0.001.

Results

Peroxynitrite modulates tyrosine phosphorylation ofsynaptophysinWe have previously demonstrated in synaptosomes thatperoxynitrite treatment, up to 0.5 mM, increased the level ofprotein tyrosine phosphorylation while at higher concentra-tions the pY signal was down-regulated. We have alsoreported that tyrosine nitration became detectable at 0.1 mMbut was predominant at 1 mM peroxynitrite and we identifiedSYP as one of the substrate nitrated by peroxynitrite (Di Stasiet al. 1999). To test the hypothesis that treatment ofsynaptosomes with peroxynitrite can also induce tyrosinephosphorylation of SYP, we immunoprecipitated SYP andthe immunocomplexes were analysed by western blot withanti-pY antibody (Fig. 1). We observed an increase in

ONOO– [mM]

Anti-NO2Y

IP SYP

Anti-pY

Anti-SYP

0 0.1 1

WB

*

*

*

Fig. 1 Peroxynitrite induces phosphorylation and nitration of SYP

tyrosine residues. Western blot analysis of SYP immunoprecipitated

from synaptosomes treated with 0.1 and 1 mM peroxynitrite. SYP

immunoprecipitates (IP SYP) were probed with the following mono-

clonal antibodies: anti-phosphotyrosine (anti-pY), anti-nitrotyrosine

(anti-NO2Y) and anti-SYP. The immunoreactive bands were detected

by chemiluminescence coupled to peroxidase activity (ECL). The

arrowheads indicate the position of SYP and the asterisks indicate the

position of IgG heavy chain.



tyrosine phosphorylation of SYP in synaptosomes treatedwith 0.1 mM peroxynitrite. When synaptosomes were treatedwith 1 mM peroxynitrite, a concentration that we previouslyreported to induce tyrosine nitration in synaptic proteins (DiStasi et al. 1999), no increase in SYP phosphorylation wasdetected, whereas, as expected, SYP was nitrated (Fig. 1).Thus, treatment of synaptosomes with peroxynitrite canmodify tyrosine residues of SYP in different ways, depend-ing on its concentration.

Stimulation of src activity by phosphorylated-synaptophysinBased on the results described above and our findings that theactivity of src, lyn and fyn kinases was modulated byperoxynitrite (Di Stasi et al. 1999; Mallozzi et al. 1999;Minetti et al. 2002), we investigated whether tyrosine-mod-ified SYP can affect src kinase activity. We performed an invitro kinase assay using the exogenous pp60src recombinantenzyme and analysed its autophosphorylation level in thepresence of SYP immunoprecipitated from untreated as wellas from 0.1 mM or 1 mM peroxynitrite-treated synapto-somes. [32P] incorporation into the band corresponding to srckinase (Fig. 2a) and the densitometric analysis of the bandsafter autoradiography (Fig. 2b) indicated that when SYP wasphosphorylated it induced an approximately twofold increasein src autophosphorylation activity. On the contrary, whenSYP was nitrated only a slight increase in src autophospho-rylation activity was observed (Fig. 2a and b). It should benoted that phosphorylation of SYP, known to be a srcsubstrate, was also enhanced (approximately threefold) by theup-regulated kinase (Fig. 2a and b).

Tyrosine-phosphorylated GST–SYP binds to and activatessrcIt is known that peptides containing specific tyrosine-phos-phorylated sequences can induce src activation in competitionwith phosphorylated regulatory tyrosine (Tyr527 for src) in thetail of the kinase (Liu et al. 1993). As the cytosolic C-terminaldomain of SYP contains putative phosphorylation sites(Fig. 3a), the stimulatory effect of tyrosine-phosphorylatedSYP on src activity could be mediated by the interaction of theC-terminal domain with the src-SH2 domain. To verify thishypothesis we expressed the SYP C-terminal domain as GST-fusion protein (GST–SYP) (Fig. 3b). Tyrosine phosphoryla-tion of GST–SYP was obtained either directly in TKB1E. coli, GST–SYP (pY)B, or in vitro by src kinase, GST–SYP(pY)src. The GST–SYP (NO2Y) was obtained by in vitrotreatment with 1 mM peroxynitrite. Figure 3b shows thewestern blot analysis of GST–SYP with anti-pY antibody(Fig. 3b, central panel) and with an anti-NO2Y antibody(Fig. 3b, right panel).

To test if the phosphorylated C-terminal domain of SYPcan bind to src, we used Sepharose-immobilised GST–SYP,GST–SYP (pY)src and GST–SYP (NO2Y) to pull-down

specific interacting proteins from rat brain synaptosomalextract. The western blot analysis with anti-src antibodyshowed that only GST–SYP (pY)src was able to pull-downsrc (Fig. 3c). We also investigated whether the enzymaticactivity of src was affected by the tyrosine-modified GST–SYP using an in vitro kinase assay. The assay was performedby incubating the GST–SYP with pp60src recombinantenzyme and enolase as exogenous substrate. As shown inFig. 3d, src kinase activity was increased by the presence ofGST–SYP (pY)src as indicated by [32P] incorporation in theelectrophoretic bands corresponding to src (autophosphory-lation) and enolase. Similar results were obtained with GST–SYP (pY)B (data not shown). It is interesting that GST–SYP(NO2Y), which does not bind to src, was also unable toinduce its activation (Fig. 3c and d).

Phosphorylated SYP binds to src through the SH2 domainIt is possible that phosphorylated SYP binds to and activatessrc through an involvement of the kinase SH2 domain. To

src

0 0.1 1

src (3U) + + + +In vitro kinase assay

IP SYP

0 0.1 10 0.1 1

SYP

SYPsrc

ONOO– [mM]

ONOO– [mM]0

1

2

3

4 (b)

(a)

[32 P

] in

corp

orat

ion

(fol

d in

crea

se)

*

***

Fig. 2 Stimulation of src kinase activity by phosphorylated SYP. (a)

Autoradiograph of in vitro kinase assay of pp60src recombinant en-

zyme (3 U) in the presence of SYP immunoprecipitated from un-

treated, 0.1 mM and 1 mM peroxynitrite-treated synaptosomes. The

[32P]-labelled proteins were resolved by SDS–PAGE (10%) and re-

vealed on dried gel by exposure to radiographic film. (b) The intensity

of the bands corresponding to src and SYP was quantified by densi-

tometric scanning of the autoradiograph shown in (a). Data are the

mean ± SD obtained from three separate experiments; *p < 0.05 and

***p < 0.001 (Student’s t-test).



verify this hypothesis, the biotin-labelled recombinant src-SH2 domain was used in a pull-down experiment withSepharose-immobilised GST–SYP, GST–SYP (pY)B, GST–SYP (pY)src and GST–SYP (NO2Y). Figure 4 shows that theSH2 domain preferentially associated with the phosphory-lated forms of GST–SYP.

We further tested the ability of native SYP purified fromsynaptosomes to bind to the src-SH2 domain. SYP waspurified from untreated and peroxynitrite-treated rat brainsynaptosomes on hydroxyapatite/celite resin (Gincel andShoshan-Barmatz 2002), as described in Appendix S1 andFig. S1. We next used purified native SYP in pull-downexperiments with the GST-src-SH2 domain and found thatGST-src-SH2 pulled down exclusively phosphorylated SYP

(Fig. 5a, upper panel). In addition, as the treatment ofsynaptosomes with 0.1 mM peroxynitrite induced anincrease in tyrosine phosphorylation, several synaptic pro-teins were pulled down by GST-src-SH2, as revealed bywestern blot analysis with anti-pY antibody (Fig. 5b, upperpanel). The GST-src-SH2 domain binds SYP from 0.1 mMperoxynitrite-treated synaptosomes as well as from untreatedsynaptosomes, indicating that a fraction of SYP is alreadytyrosine-phosphorylated but that the phosphorylation levelincreases following peroxynitrite treatment. Interestingly,although in synaptosomes treated with 1 mM peroxynitritesome tyrosine-phosphorylated proteins are still present andpulled down by the GST-src-SH2 domain, the bindingbetween SYP and GST-src-SH2 was almost completely

src

SYP

Pull-down assay

66

45

29

kDa

Enolasesrc

SYP

In vitro kinase assay + + + + src (1U)

(a)

(b)

(c) (d)

Fig. 3 Tyrosine-phosphorylated GST–SYP binds and activates src.

(a) Schematic representation of SYP structure and its C-terminal do-

main sequence (amino acids 219–308) showing in grey the nine

copies of the tyrosine-rich pentapeptide. (b) The C-terminal domain of

SYP expressed as GST-fusion protein and GST alone stained with

Red Ponceau (left panel) or probed with anti-pY (central panel) and

with anti-NO2Y (right panel) antibodies. The molecular mass stan-

dards in kDa are indicated on the left. (c) Pull-down assay with

Sepharose-immobilised GST–SYP, GST–SYP (pY)src and GST–SYP

(NO2Y) from rat brain synaptosomal extract. The presence of src in the

pull-down was revealed by western blot with anti-src antibody and the

immunoreactive bands were detected by chemiluminescence coupled

to peroxidase activity (ECL). The amount of SYP (input) in the pull-

down was evaluated by anti-SYP antibody. The molecular mass

standards in kDa are indicated on the left. (d) Autoradiograph of in vitro

kinase assay of recombinant src (1 U) performed in the presence of

GST–SYP, GST–SYP (pY)src and GST–SYP (NO2Y). Enolase was

added to the reaction mixture as exogenous substrate. The [32P]-la-

belled proteins were resolved on 10% SDS–PAGE and shown on dried

gel by exposure to X-ray film. The results shown are representative of

three independent experiments.



abolished (Fig. 5b, lower panel). Moreover, other synapticproteins that interact with src-SH2, such as synapsin I, werenot affected by peroxynitrite treatment (Fig. 5b, lower panel).

The specificity of the interaction between phosphorylatedSYP and the src-SH2 domain was further demonstrated byusing a different SH2 domain, such as that of the 85 kDasubunit of Phosphoinositide 3-kinase. As shown in Fig. 5c(upper panel), the GST-P85-SH2 pulled down its tyrosine-phosphorylated interactors from 0.1 mM peroxynitrite-treated synaptosomes, but did not pull-down SYP in anyconditions tested (Fig. 5c, lower panel).

Mapping the tyrosine residues of SYP modified byperoxynitrite treatmentThe analysis of modified tyrosine residues of GST–SYPwas performed by MS on digest mixture coming from AspNand chymotrypsin: the second enzyme was particularly usefulfor nanospray MS/MS analysis thanks to the formation ofpeptides that are bigger than those obtained from AspNtreatment and contains some basic residues capable to improvethe ionisation and increase peptide charge. MALDI–ToFanalysis of peptide mixture coming from GST–SYP (pY)Bafter AspN digestion has shown the presence of two phos-phorylated sequences: aa 261–271 and aa 272–288 (seeFig. S2a and b), each containing two tyrosine residues. Torecognisewhichof the two tyrosine residues is phosphorylated,tandem MS spectra of peptides, obtained after chymotrypsin

SH2

Pull-down assay

SYP WB anti-SYP

Avidin-biotin complex

SH2 [ M]

Fig. 4 Pull-down assay of tyrosine-modified GST–SYP with biotin-la-

belled src-SH2 domain. Pull-down assay with Sepharose-immobilised

GST–SYP, GST–SYP (pY)src, GST–SYP (pY)B and GST–SYP

(NO2Y) incubated in the presence of biotin-labelled src-SH2 domain at

0.2 and 2 lM. The bound SH2 in the pull-down samples was revealed

by western blot analysis using peroxidase-conjugated avidin complex

with biotin followed by ECL procedure. The results shown are repre-

sentative of three independent experiments.

(a) (b) (c)

Input

Fig. 5 Tyrosine-phosphorylated SYP specifically binds the src-SH2

domain. (a) Western blot of the pull-downs obtained by incubating

Sepharose-immobilised GST-src-SH2 with native SYP purified using

the hydroxyapatite/celite column from untreated, 0.1 mM and 1 mM

peroxynitrite-treated synaptosomes. The presence of bound SYP in

pull-down samples was revealed by anti-SYP antibody (upper panel).

Central panel: western blot of pull-down samples with anti-GST

antibody. Lower panel (input): control of the native SYP used in the

pull-downs revealed with anti-SYP antibody. (b) Western blot of the

pull-downs obtained by incubating Sepharose-immobilised GST-src-

SH2 with solubilised peroxynitrite-treated synaptosomes at the indi-

cated concentrations. The samples were probed with the following

antibodies: anti-pY, anti-SYP, anti-synapsin I (SYN I) and anti-GST.

(c) Western blot of the pull-downs obtained by incubating Sepharose-

immobilised GST-p85-SH2 with solubilised peroxynitrite-treated syn-

aptosomes at the indicated concentrations. As described in (b), the

pull-down samples were probed with anti-pY, anti-SYP and anti-GST

antibodies. The immunoreactive bands in all experiments were

detected by ECL procedure. The results shown are representative of

three independent experiments.



treatment, were acquired allowing to assign the phosphoryla-tion site on Tyr263 (Fig. 6a) and on Tyr273 (Fig. 6b).

To identify tyrosine residues nitrated by peroxynitrite,GST–SYP (NO2Y) and GST–SYP peptides mixture wasanalysed by MALDI–ToF analysis (following AspN diges-tion) or by LC–MS/MS analysis (following chymotrypsindigestion). MALDI–ToF analysis restricts the nitration to thepeptide (aa 247–260) (see Fig. S3a and b) and MS/MSanalysis enables the assignment of the nitration to the Tyr250(Fig. 6c). The summary of the location of these modificationsis displayed in Fig. 6d.

Discussion

The results presented in this study indicate that in peroxy-nitrite-treated synaptosomes tyrosine residues in the cyto-

plasmic C-terminal tail of SYP are either phosphorylated ornitrated, depending on the concentration of peroxynitrite.When phosphorylated, SYP activates src kinase by specifi-cally interacting with the src-SH2 domain. In contrast, whenit is nitrated, SYP fails to interact with the src-SH2 domainand to stimulate src kinase activity. Using MS, we mappedthe SYP C-terminal tail tyrosine residues modified byperoxynitrite and identified one nitration site at Tyr250 andtwo phosphorylation sites at Tyr260 and Tyr273. Ourfindings suggest that modifications of SYP specific tyrosineresidues may differently affect the binding properties of SYPand highlight the relevance of SYP as one of peroxynitritetargets, generated at the nerve terminals in pathophysio-logical conditions.

The SV protein SYP is known to be phosphorylated by srckinase both in vitro and in SVs (Barnekow et al. 1990;

(a)

(b)

(c)

(d)

Fig. 6 Localization of phosphorylated and

nitrated tyrosine residues by MS/MS anal-

ysis. LC–MS/MS analysis of peptides

obtained from chymotrypsin has been

performed: tandem mass spectra of the

precursor ions [MH2]2+ = 1074.2 (a),

[MH2]2+ = 1195.3 (b) and [MH3]3+ = 793.14

(c) are displayed with the corresponding

sequences. Peaks matching the b and y

series are shown in the spectra and in the

sequences: the stars indicate the ions with

the addition of the mass increase corre-

sponding to the modification (+ 80 Da for

the phosphotyrosine in panels a and b, and

+ 45 Da for the nitrotyrosine in panel c). pY

indicates the distance between two peaks

corresponding to a phosphotyrosine (pan-

els a and b) while nY, in panel (c), points to

y1 + 1 ion matching to the nitrotyrosine.

Panel (d) displays the sequence analysed

by MS/MS analysis and in bold are

highlighted the residues carrying the modi-

fications.



Onofri et al. 1997). Here, we found that peroxynitritetreatment of synaptosomes induces SYP phosphorylationon tyrosine, which in turn stimulates src activity. In addition,we found that the tyrosine-rich C-terminal tail of SYP is thetarget for phosphorylation. Indeed, the recombinant GST-C-terminal tail of SYP, when phosphorylated on tyrosine,stimulates src activity similarly to the phosphorylated SYPpurified from peroxynitrite-treated synaptosomes. Thisallows the use of the recombinant protein to investigatehow specific tyrosine modifications affect the interaction ofSYP with its binding partners.

The finding that the phosphorylated SYP increases srckinase activity by interacting with the src-SH2 domain fitsvery well with the molecular model suggested for src familykinase activation. The kinases of the src family share a highdegree of structural similarity, being organised in modulardomains: SH3, SH2, catalytic and unique. The SH3 and SH2domains play a role in src regulation, both being required tokeep the kinases in an inactive state and to target the enzymeto specific substrates by modulating protein–protein interac-tions. The catalytic activity of src kinases is regulated by thephosphorylation of two tyrosine residues in contrasting ways.The autophosphorylation of Tyr416, located inside thecatalytic domain, correlates with the enzyme activation,while the phosphorylation of the C-terminal tyrosine(Tyr527) gives rise to an inactive form of the enzyme byinteracting with the SH2 domain (Thomas and Brugge 1997).As this interaction is relatively weak, exogenous ligands canefficiently compete with SH2, promoting kinase activation.According to this model, upon phosphorylation by src, SYPbehaves like a src-SH2 domain ligand, thus stimulating srcautophosphorylation and activity.

MALDI–ToF analysis of the peptides coming from therecombinant C-terminal tail of phosphorylated SYP identi-fied two phosphorylated sequences: aa 261–271 and aa 272–288. The phosphatase treatment confirm the presence of onephosphorylated residue for each peptide and tandem MSassigns the modification to the residues Tyr263 and Tyr273.However, further studies should be carried out to elucidate ifboth or only one phosphorylated tyrosine specificallyinteracts with src and stimulates its activity.

At the nerve terminal, several proteins are substrates of src(Benfenati et al. 1999; Valtorta et al. 2004), includingsynapsin I, which is phosphorylated at a single residue(Tyr301) and interacts with the SH2 domain, contributing tosrc activation (Onofri et al. 2007). However, we found thatperoxynitrite does not affect synapsin I phosphorylationlevels, as indicated by the fact that equal amounts ofphosphorylated synapsin I were pulled down by the src-SH2domain from untreated and from peroxynitrite-treated syn-aptosomes. In addition, tubulin is another protein known tointeract with and be phosphorylated by src (Matten et al.1990), but in our experimental conditions the GST-src-SH2failed to pull-down tubulin from peroxynitrite-treated syn-

aptosomes (our unpublished observation). These resultssuggest that specific synaptic proteins, such as SYP, areinvolved in the modifications induced by peroxynitritegenerated at the nerve terminal in conditions that promoteoxidative stress, including ischaemic conditions or neurode-generative disorders (Ischiropoulos and Beckman 2003;Casoni et al. 2005). We have also reported that excitotoxicityand oxidative stress, induced by intrastriatal injection ofquinolinic acid as a model of Huntington’s disease, involvedthe modulation of src and lyn activity (Metere et al. 2006).

It has been reported that src activity and src-SYPinteractions are increased in vivo in learning and that tyrosinephosphorylation of SYP is enhanced during synaptic stim-ulation as is known to occur during the long-term potenti-ation (Zhao et al. 2000). In this model, the phosphorylatedSYP could recruit src, thereby stimulating a further increasein kinase activity. Interestingly, the interaction of SYP withdynamin I, a synaptic protein involved in endocytosis, maybe regulated by SYP phosphorylation, given that thetyrosine-based pentapeptide motifs on the SYP C-terminalare the binding sites for dynamin I (Daly and Ziff 2002).

As well as being tyrosine-phosphorylated, SYP is one ofthe major pre-synaptic proteins nitrated by peroxynitrite (DiStasi et al. 1999). In a previous work, we demonstrated thatNO2Y-containing peptides were able to activate the tyrosinekinase lyn similarly to phosphopeptides through an SH2-dependent mechanism (Mallozzi et al. 2001). Interestingly,here we found that nitrated SYP, unlike phosphorylated SYP,does not interact with the src-SH2 domain and does notstimulate src activity. This finding may be explained by theMS/MS analysis results showing that SYP is nitrated onTyr250 located in a sequence different from those containingtyrosine-phosphorylated residues. This suggests that the ninerepeats in the SYP C-terminal domain are not equivalent andthat the residues adjacent to the consensus YG(P/Q)QG arecritical for both SYP tyrosine modifications and SYPinteractions with its binding partners.

Recently, Felkl and Leube (2008) using fluorescenceresonance energy transfer technique, reported that C-terminalSYP is recognised by the src-SH2 domain and that deletion ofall tyrosine residues abolished this interaction. It is of interestto note that the exposed cytoplasmic C-terminus of SYP isinvolved in the binding with various nerve terminal pro-teins, including v-soluble N-ethylmaleimide sensitive fusionproteins attachment receptor VAMP2, vesicular proton pumpV-ATPase, myosin V, c-adaptin and dynamin I. Therefore,phosphorylation and/or nitration of different tyrosine residuesof the SYP C-terminal tail could regulate these associationsby modulating mutual affinities of the various bindingpartners. In this contest it has been reported that in amyloidb1–40-infused rats SYP was nitrated and phosphorylated, andits ability to recruit VAMP2 modified (Tran et al. 2003).

The reactive oxygen and nitrogen species can be criticalfor excitatory synaptic activity and, as we previously



reported, peroxynitrite can significantly stimulate vesicleexocytosis and induce endogenous glutamate release fromsynaptosomes (Di Stasi et al. 2002). Increasing experimentalfindings suggest that tyrosine nitration may be involveddirectly or indirectly in the regulation of signalling proteins(Monteiro et al. 2008). In this respect, identification ofpotential redox-modified targets could have a pathophysio-logical relevance in neurodegenerative diseases.

Acknowledgements

This work was supported in part by the Italian Ministry of Health,

Istituto Superiore di Sanita: Grants SLA5-Art. 533 and IS-NEURO

ex 56/05/20. We thank Dr F. Malchiodi-Albedi for helpful

comments on the manuscript.

Supporting information

Additional supporting information may be found in the online

version of this article:

Appendix S1 Supplementary Materials and methods.

Appendix S2 Legends to Supplementary Figures.

Fig. S1 Purification of SYP from synaptosomes using a

hydroxyapatite/celite column.

Fig. S2 MALDI–ToF analysis of GST–SYP (pY)B. MALDI–

ToF-MS spectra of the peptide mixtures derived from GST-SYP

(pY)B (A and B) after in-gel digestion by AspN and phosphatase

treatment (C and D).

Fig. S3 MALDI–ToF analysis of GST–SYP (NO2Y). MALDI–

ToF-MS spectra of the peptide mixtures derived from GST-SYP (A)

and from GST-SYP (NO2Y) (B) after in-gel digestion by AspN.

As a service to our authors and readers, this journal provides

supporting information supplied by the authors. Such materials are

peer-reviewed and may be re-organized for online delivery, but are

not copy-edited or typeset. Technical support issues arising from

supporting information (other than missing files) should be

addressed to the authors.

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